U.S. patent application number 13/092667 was filed with the patent office on 2011-11-10 for arrestin biosensor.
Invention is credited to Alexandre Beautrait, Michel Bouvier, Pascale Charest, Christian Le Gouill.
Application Number | 20110275134 13/092667 |
Document ID | / |
Family ID | 44902190 |
Filed Date | 2011-11-10 |
United States Patent
Application |
20110275134 |
Kind Code |
A1 |
Bouvier; Michel ; et
al. |
November 10, 2011 |
Arrestin Biosensor
Abstract
The present invention relates to a novel biosensor. A resonance
energy transfter (RET) biosensor comprising a beta(.beta.)-arrestin
tagged with a first and a second chromophore, wherein said first
chromophore is a fluorophore and said second chromophore is a
fluorophore or a bioluminophore is described.
Inventors: |
Bouvier; Michel; (Montreal,
CA) ; Charest; Pascale; (San Diego, CA) ; Le
Gouill; Christian; (Montreal, CA) ; Beautrait;
Alexandre; (Montreal, CA) |
Family ID: |
44902190 |
Appl. No.: |
13/092667 |
Filed: |
April 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11579482 |
Apr 28, 2008 |
7932080 |
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PCT/CA2005/000695 |
May 4, 2005 |
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13092667 |
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60567454 |
May 4, 2004 |
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Current U.S.
Class: |
435/188 ;
530/402 |
Current CPC
Class: |
G01N 33/542 20130101;
G01N 33/582 20130101; G01N 2333/726 20130101; G01N 33/533 20130101;
G01N 2333/4704 20130101 |
Class at
Publication: |
435/188 ;
530/402 |
International
Class: |
C12N 9/96 20060101
C12N009/96; C07K 19/00 20060101 C07K019/00; C07K 17/02 20060101
C07K017/02 |
Claims
1. A resonance energy transfter (RET) biosensor comprising an
arrestin tagged with a first and a second chromophore, wherein said
first chromophore is a fluorophore and said second chromophore is a
fluorophore or a bioluminophore.
2. The biosensor according to claim 1, wherein said arrestin is
.beta.-arrestin-1 (arrestin-2).
3. The biosensor according to claim 1, wherein said arrestin is
.beta.-arrestin-2 (arrestin-3).
4. The biosensor according to claim 1, wherein said arrestin is
arrestin-1 or arrestin-4.
5. The biosensor according to claim 1, wherein said second
chromophore is a bioluminophore and the RET is bioluminescence
resonance energy transfter (BRET).
6. The biosensor according to claim 5, wherein said bioluminophore
is Renilla luciferase or a mutant form of Renilla luciferase.
7. The biosensor according to claim 6, wherein said bioluminophore
is RlucII (A55T/C124A/M185V).
8. A biosensor as defined in claim 1, wherein said fluorophore is a
fluorescent protein.
9. The biosensor as defined in claim 8, wherein said fluorophore is
green fluorescent protein or a variant thereof.
10. The biosensor of claim 9, wherein said fluorophore is YFP,
mAmetrine, cyan fluorescent protein (CFP), or GFP10.
11. A biosensor as defined in claim 5, which is selected from the
group consisting of: Luc-.beta.-arr-YFP, YFP-.beta.arr-Luc,
Luc-.beta.-arr(3A)-YFP, Luc-.beta.-arr(IV)-YFP,
Luc-.beta.arr(R169E)-YFP, Ametrine-h.beta.ar1-RlucII,
Ametrine-h.beta.arr2-RlucII, CFP-h.beta.arr1-RlucII,
CFP-h.beta.arr2-RlucII, GFP.sup.10-h.beta.arr1-RlucII, and
GFP.sup.10-h.beta.arr2-RlucII.
12. The biosensor as defined in claim 5, wherein the fluorophore is
at the N-terminus of the arrestin and the bioluminophore is at the
C-terminus of the arrestin.
13. The biosensor as defined in claim 5, wherein the fluorophore is
at the C-terminus of the arrestin and the bioluminophore is at the
N-terminus of the arrestin.
14. The biosensor of claim 5, wherein one of the fluorophore or
bioluminophore is linked to a position between the C and
N-termini.
15. The biosensor as defined in claim 14, wherein the
bioluminophore is linked to the C-terminus of the arrestin and the
fluorophore is linked internally to the arrestin.
16. The biosensor of claim 15, wherein the fluorophore is YFP,
mAmetrine, cyan fluorescent protein (CFP), or GFP10, and the
bioluminophore is Renilla luciferase or a mutant form of Renilla
luciferase.
17. The biosensor according to claim 1, wherein both said first and
said second chromophores are fluorophores and the RET is
fluorescence resonance energy transfer (FRET).
18. The biosensor of claim 17, wherein the fluorophore donor is CFP
or a variant thereof, and the fluorophore acceptor is a YFP or a
variant thereof.
19. The biosensor of claim 18, wherein the YFP is a non-ciruclarly
permuted sYFP2 or a circularly permuted sYFP2.
Description
TECHNICAL FIELD
[0001] The invention relates to a novel biosensor and method
suitable for monitoring activation of receptors and signaling
molecules. More specifically, the invention concerns the use of a
modified arrestin as a biosensor to monitor the activation state of
receptors, such as G protein-coupled receptors (GPCR).
Advantageously, the biosensor and method of the present invention
allow for a highly sensitive and quantitative assay that can be
used in large-scale screening analyses.
BACKGROUND OF THE INVENTION
[0002] The largest class of cell surface receptors in mammalian
genomes is the superfamily of G protein-coupled receptors (GPCRs).
GPCRs are proteins that span the membrane of a cell and relay the
information provided by numerous ligands, e.g. hormones and
neurotransmitters, into intracellular signalling pathways. GPCRs
are thus the targets of many clinically important drugs, with
approximately half of all current prescription drugs acting through
GPCRs (Drews J (1996) Genomic sciences and the medicine of
tomorrow. Nat Biotechnol 14: 1516-1518). Examples of GPCRs are many
and include beta-2 adrenergic receptor (.beta.2-AR), Frizzled 4
(Fz4), V2-vasopressin receptor (V2R), V1a vasopressin receptor
(V1aR), .delta.-opioid receptor (.delta.-OR), platelet-activating
factor receptor (PAFR), CC chemokine receptor type 5 (CCR5), and
angiotensin receptor type 1a (AT1 aR).
[0003] GRCRs relay the information encoded by the ligand (e.g.
hormones and neurotransmitters) through the activation of G
proteins and intracellular effector molecules. G proteins are
heterotrimeric proteins, consisting of an alpha, a beta, and a
gamma subunit. The three G-subunits are non-covalently bound
together and the G protein as a whole binds to the inside surface
of the cell membrane and associates with the GPCR. Starting in such
conformation, the G-alpha subunit is complexed to GDP (guanosine
diphosphate). When a ligand binds to a domain of the GPCR
accessible from the outside of the cell membrane, a conformational
change in the GPCR occurs, which in turn prompts the exchange of
the GDP for a molecule of guanosine triphosphoate (GTP) on the
G-alpha subunit, and activates the G-protein. The G-protein's
.alpha. subunit, together with the bound GTP, can then dissociate
from the .beta. and .gamma. subunits to further affect
intracellular signaling proteins or target functional proteins
directly, depending on the .alpha. subunit type (e.g. G.alpha.s,
G.alpha.i/o, G.alpha.q/11, G.alpha.12/13).
[0004] In order to turn off this response by GPCRs to stimulus, or
adapt to a persistent stimulus, the activated GPCRs are
inactivated. This inactivation may be achieved, in part, by the
binding of a soluble protein, .beta.-arrestin (.beta.-arr), which
uncouples the receptor from the downstream G protein after the
receptor is phosphorylated by a G protein-coupled receptor kinase
(GRK). More specifically, through their binding to
agonist-occupied, GRK-phosphorylated receptors, .beta.-arrs prevent
further coupling to G proteins and promote GPCR endocytosis, thus
leading to decreased signalling efficacy.
[0005] Despite our growing understanding of the diversity in GPCR
signaling mechanisms, drug efficacy is often defined only in terms
of the regulation of the classical G protein signaling. Within this
framework, agonists are defined as drugs that stabilize an active
receptor conformation that induces G protein activation, whereas
inverse agonists favor an inactive receptor state that reduces
spontaneous G protein signaling. The question arises as to whether
this paradigm may be transferred to drug effects generated through
the formation of metastable complexes involving scaffolding
proteins such as .beta.-arr. Because all studies describing
.beta.-arr-mediated MAPK signalling have concentrated on agonist
drugs, little is known of how ligands that are commonly classified
as inverse agonists may regulate the scaffold assembly that is
crucial for such signalling.
[0006] In one study (Azzi et al, 2003), this question was addressed
by assessing whether .beta.-adrenergic receptor (.beta.2AR) and V2
vasopressin receptor (V2R) ligands with proven inverse efficacy on
adenylyl cyclase (AC) activity could also regulate MAPK activation
via receptor-mediated scaffold formation. It was found that,
despite being inverse agonists in the AC pathway, the .beta.2AR
(ICI118551 and propranolol) and V2R(SR121463A) induced the
recruitment of .beta.-arr leading to the activation of the ERK
cascade. Such observations indicate that the same drug acting on a
unique receptor can have opposite efficacies depending on the
signaling pathway considered.
[0007] The above study relied on the use of a bimolecular
bioluminescence resonance energy transfer (BRET) assay. It was used
to assess .beta.-arrestin recruitment to .beta.2AR or V2R. Fusion
proteins consisting of GFP10 variant (GFP) covalently attached to
the carboxyl tail of the receptor of interest (.beta.2AR-GFP;
V2R-GFP) were co-expressed with .beta.-arrestin 2 fused at its
carboxyl terminus to Rluc (.beta.-arrestin-Rluc). After incubation
of the transfected cells with different ligands, coelenterazine
400a (Perkin-Elmer, Wellesley, Mass., USA) was added and readings
were collected using a modified top-count apparatus (BRETCount,
Packard) that allows the sequential integration of the signals
detected at 370-450 nm and 500-530 nm. The BRET signal was
determined by calculating the ratio of the light emitted by the
Receptor-GFP (500-530 nm) over the light emitted by the
.beta.-arrestin2-Rluc (370-450 nm). The values were corrected by
subtracting the background signal detected when the
.beta.-arrestin2-Rluc construct was expressed alone.
[0008] While the results elicited from the above study were
instructive, a necessary feature involved the construction of
fusion proteins that included the receptors of interest. Ideally, a
method could be devised in which receptor activation might be
observed without first having to modify the receptors that are to
be studied. Other features of such a method that would make it
highly desirable for research and development endeavors include the
following: (1) a high level of sensitivity; (2) an ability to
provide quantitative results; (3) adaptability for use in large
scale screening analyses; (4) an assay that requires the expression
of a single recombinant construct; and (5) a biosensor based on an
intramolecular RET signal.
[0009] Resonance energy transfer (abbreviated RET, and also
referred to as Forster resonance energy transfer), is a mechanism
describing energy transfer between two chromophores, having
overlapping emission/absoprtion spectra. When the two chromophores
(the "donor" and the "acceptor"), are within 10-100 .ANG. of one
another and their transition dipoles are appropriately oriented,
the donor chromophore is able to transfer its excited-state energy
to the acceptor chromophore through nonradiative dipole-dipole
coupling. When both chromophores are fluorescent, the term
typically used is "fluorescence resonance energy transfer"
(abbreviated FRET). In bioluminescence resonance energy transfer
(BRET), the donor chromophore of the RET pair, rather than being a
fluorophore, is a bioluminescent molecule, typically luciferase. In
the presence of a substrate, bioluminescence from the donor excites
the acceptor fluorophore through the same Forster resonance energy
transfer mechanism described above (Xu, Y. et al., PNAS, 96:151-156
(1999)).
[0010] There is a need for a simpler method to measure receptor
activity in living cells. The present invention seeks to meet this
and related needs.
SUMMARY OF THE INVENTION
[0011] In accordance with the present invention, a resonance energy
transfter (RET) biosensor comprising an arrestin tagged with a
first and a second chromophore, wherein said first chromophore is a
fluorophore and said second chromophore is a fluorophore or a
bioluminophore, is provided.
[0012] Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of preferred embodiments thereof, given
by way of example only with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1: Double-brilliance .beta.-arr. Schematic diagram
illustrating how agonist-promoted conformational rearrangement of
.beta.-arr can be measured as changes in BRET using
double-brilliance .beta.-arr. Luc and YFP are represented by
cylinders proportional to their sizes, but their real orientation
is unknown.
[0014] FIG. 2: Functionality of double-brilliance .beta.-arr.
HEK293 (A-C) or COS (D) cells were transiently transfected with the
indicated plasmids. (A) Cells incubated or not in the presence of
saturating concentrations of specific agonists (.beta.2-AR, 10 mM
isoproterenol (ISO); V2R, 1 mM arginine vasopressin (AVP)).
Localization of Luc-.beta.-arr-YFP and Myc-tagged receptors was
analysed by confocal fluorescence microscopy. (B) Agonist-induced
recruitment of .beta.-arr measured using intermolecular BRET2.
t1/2=half-time of maximal .beta.-arr recruitment. (C)
Dose-dependent recruitment of .beta.-arr to the receptors measured
in intermolecular BRET2 following 2 min stimulation with the
agonist. EC50=concentration of agonist producing half-maximal
.beta.-arr recruitment. (D) Cells treated or not for 15 min with
the specific agonists at 37.degree. C. and cell-surface receptor
levels measured by enzyme-linked immunosorbent assay (ELISA).
Receptor endocytosis is defined as the loss of cell-surface
immunoreactivity and is expressed as a percentage of total
immunoreactivity measured under basal conditions. Expression levels
of .beta.-arr were controlled using western blot (data not shown).
Data are the mean.+-.s.e.m. of at least three independent
experiments. *P<0.05 between treatment and each individual
control condition. Mock, non-transfected cells.
[0015] FIG. 3: AVP-induced conformational change of .beta.-arr
monitored by intramolecular BRET1. HEK293 cells were transfected
with the indicated plasmids and BRET was measured at 25.degree. C.
in the presence of coelenterazine h. (A) Specificity of
agonist-induced .beta.-arr intramolecular BRET1. (B) Real-time BRET
measurements of the agonist-induced .beta.-arr conformational
change. t1/2=half-time of maximal conformational change of
.beta.-arr. (C) Dose-dependent agonist-promoted increase of
.beta.-arr intramolecular BRET1. Cells were stimulated with
increasing concentrations of AVP for 4 min. EC50=concentration of
AVP producing half-maximal conformational change of .beta.-arr.
Data are the mean.+-.s.e.m. of at least three independent
experiments. *P<0.01 between treated and control condition.
[0016] FIG. 4: Agonist-promoted conformational change of a
phosphate insensitive .beta.arrestin mutant. HEK293 cells were
transfected with V2R and either Luc-.beta.arr-YFP or
Luc-.beta.arr(R169E)-YFP. Cells were stimulated or not for 10 min
with 1 .mu.M AVP prior the addition of 5 .mu.M coelenterazine h
(Molecular Probe) and performing the intramolecular BRET1
measurements using a Multilabel Reader Mithras LB 940 (Berthold
Technologies). The BRET signal was determined by calculating the
ratio of the light emitted by YFP over the light emitted by Luc
following the addition of coelenterazine h. The values were
corrected by subtracting the background BRET signals detected when
Luc-.beta.arr was expressed alone. Inset, AVP-induced BRET
increase. Data represent the mean.+-.SEM of three independent
experiments. * indicates p<0.02 between treatment and each
individual control condition.
[0017] FIG. 5: Double-brilliance .beta.-arr monitors the activation
of many GPCRs. HEK293 cells were transfected with
Luc-.beta.-arr-YFP and either pcDNA3.1 or plasmids encoding the
indicated receptors. (A) Agonist-induced translocation of
Luc-.beta.-arr-YFP measured following treatment with 1 mM of the
specific agonists (.beta.2-AR, ISO; V1 aR, AVP; .delta.-OR, SNC80;
PAFR, PAF; CCR5, hRANTES; AT1aR, angiotensin II). (B)
Agonist-induced conformational change of Luc-.beta.-arr-YFP
measured following 10 min stimulation with the specific agonists
mentioned in (A). BRET1 was measured using a Multilabel Reader
Mithras LB 940 (Berthold Technologies). The BRET signal was
determined by calculating the ratio of the light emitted by YFP
over the light emitted by Luc following the addition of
coelenterazine h. Data are the mean.+-.s.e.m. of three independent
experiments. *P<0.05 between treatment and each individual
control condition.
[0018] FIG. 6: Agonist-promoted conformational change of
constitutively activated .beta.arrestin mutants. HEK293 cells were
transfected with V2R and either Luc-.beta.arr-YFP or Luc-.beta.arr
(3A)-YFP or Luc-.beta.arr (IV)-YFP. Cells were stimulated or not
for 10 min with 1 .mu.M AVP prior to the addition of 5 .mu.M
coelenterazine h and performing the BRET measurements as described
in the previous figure. Inset, AVP-induced BRET increase. The BRET
signal was determined by calculating the ratio of the light emitted
by YFP over the light emitted by Luc following the addition of
coelenterazine h. The values were corrected by subtracting the
background BRET signals detected when Luc-.beta.arr was expressed
alone. Data represent the mean.+-.SEM of two independent
experiments. * indicates p<0.05 between treatment and each
individual control condition.
[0019] FIG. 7: Conformational change of .beta.arrestin induced by
ligands of different efficacies. HEK293 cells transiently
co-expressing the V2R and Luc-.beta.arr-YFP were subjected to
real-time BRET measurements in the presence or absence of two
different V2R ligands. The basal BRET signals were subtracted from
each condition to express the data as ligand-induced BRET increase.
The figure shows the detection of conformational changes of
Luc-.beta.arr-YFP in time, reflected by the increase in BRET
signal, as induced by the V2R agonist AVP or the inverse agonist
SR121463. No BRET increase was observed when cells were incubated
in the presence of the carrier alone (non-stimulated). The fact
that the observed increase in BRET signal induced by SR121463 is
significantly lower than that induced by AVP treatment can be
correlated with the smaller SR121463-mediated recruitment of
.beta.arrestin to the V2R when compared to AVP, as reported
previously (Azzi et al, 2003).
[0020] FIG. 8: .beta.arrestin-dependant endocytosis beyond GPCRs.
(A) Endocytosis of the receptor Frizzled 4 (Fz4) stimulated by
Wnt5a is orchestrated by .beta.arrestin 2, in a manner that is
dependent upon the phosphorylation of the adaptor protein
Dishevelled 2 (Dv12) by protein kinase C (PKC). (B) Endocytosis of
the RII and RIII receptor subtypes of TGF-.beta.1 is orchestrated
by .beta.arrestin 2, and facilitated by the phosphorylation of RIII
by RII. (C) Endocytosis of the IGF1 receptor is orchestrated by
.beta.arrestin (Modified from Lefkowitz & Whalen, 2004.).
[0021] FIG. 9: Characterization of BRET2-.beta.Arrestin
double-brilliance sensors. (A) Structure and activation: BRET1 and
BRET2-.beta.Arrestin double brilliance (db) sensors are
unimolecular with BRET tags in N- and C-terminus of a central
.beta.Arrestin core. The linkers separating the BRET1 and BRET2
tags from .beta.Arrestin differ in both length and composition. For
the BRET1 sensor the structure is: BRET donor
(Rluc)-Linker1-.beta.Arrestin-Linker2-BRET1 acceptor (YFP) and for
the BRET2 sensors: Structure: BRET2 acceptor (sCFP3A, mAmetrine or
GFP10)-Linker3-.beta.Arrestin- Linker4- BRET2 donor (RlucII). For
BRET1, the Rluc substrate is coelenterazine H, whereas for BRET2,
the Rluc substrate is deep-blue coelentrazine. All versions of the
.beta.Arrestin1 and 2 db are conformational sensors. However,
following GPCR activation by an agonist (illustrated as a
triangle), changes in .beta.Arrestin conformation lead to a
decreased BRET signal for the BRET2 sensors while it leads to an
increased BRET signal with the BRET1 sensor configuration (see
FIGS. 1-7). (B) Kinetics and dose-responses measured in BRET2 with
the BRET2-.beta.ARR1 and 2 db sensors, in response to V2R
activation by its agonist AVP: at 100 nM for the kinetics or at
increasing concentrations of AVP for dose-response experiments. (C)
.beta.ARR db sensor to characterize ligands of different
efficacies. Hek293 cells transiently expressing both AT1aR and
GFP10-.beta.arr1-RlucII db sensor, were stimulated with a full
(AngII) or partial agonists and responses were evaluated as a BRET2
signal modulation. a) Dose-dependent ligand-promoted decrease of
.beta.arrestin intramolecular BRET2 signal after a 25 min
stimulation. Data are the mean+/- S.E.M. of 3 independent
experiments. b) Agonist-promoted BRET changes. Cells were treated
for 25 min with 1 .mu.M AngII or 10 .mu.M of the partial agonists.
Data represent mean+/- S.E.M. of 4 independent experiments. One-way
ANOVA followed by Tukey's multiple comparison post-hoc test (AngII
as reference) was used to assess statistical significance. *,
p<0.05, ***, p<0.001. AngII=Angiotensin 2 octapeptide, SVdF:
AngII analog with Sar.sub.1,Val.sub.5,D-Phe.sub.8 substitutions at
the indicated amino acid positions in the octapeptide, SII: AngII
analog with Sar.sub.1,Ile.sub.4,Ile.sub.8 substitutions at the
indicated amino acid positions in the octapeptide, SBpA: AngII
analog with Sar.sub.1,Bpa.sub.8 substitutions at the indicated
amino acid positions in the octapeptide, SIVI: AngII analog with
Sar.sub.1,Ile.sub.s substitutions at the indicated amino acid
positions in the octapeptide, DVG: AngII analog with
Asp.sub.1,Val.sub.5,Gly.sub.8 substitutions at the indicated amino
acid positions in the octapeptide.
[0022] FIG. 10: Z'-factor evaluation for both BRET1- and
BRET2-.beta.Arrestin sensors. HEK293 cells transiently expressing
both V2R and the double-brilliance sensor, were exposed to either
100 nM AVP or a control vehicle, for 25-35 min. BRET ratios are
represented for each individual well of a 96-well plate. Z'-factors
were calculated as described in (Ji-Hu Zhang et al. 1999 J Biomol
Screen, 4; 67). A Z'-factor between 0.4 and 1 is considered a
robust assay.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0023] Unless otherwise defined, the terms used in the present
description have the meanings that would be understood by a person
of skill in the art.
[0024] Ligand: A molecule which may be but is not restricted to a
hormone, neurotransmitor, chemical compound, drug, or diagnostic
agent that binds to a receptor and has an agonistic, inverse
agonistic, antagonistic or allosteric effect on the receptor.
Ligands may be further classified as follows (for a more detailed
summary, see Wilson, Keith et al. (Eds.), Principles and Techniques
of Biochemistry and Molecular Biology, 7th Edition (2010), Chapter
17, incorporated by reference herein): [0025] a) Agonist: a ligand
that has the same or similar effect as a hormone, neurotransmitter
or signaling molecule or a group of hormones, neurotransmitters or
signaling molecules activating a receptor, by binding to the same
natural receptor. A partial agonist is a type of agonist that with
lower intrinsic activity than a full agonist and that produces a
lower maximum effect. Examples of agonists include: [0026] i.
Angiotensin II: The active form of angiotensin. An octapeptide
found in blood, it is synthesised from angiotensin I and quickly
destroyed. Angiotensin II causes profound vasoconstriction with
resulting increase in blood pressure. It is an agonist of the
angiotensin receptor. [0027] ii. AVP: arginine vasopressin,
vasopressin containing arginine, as that from most mammals,
including man. This hormone controls water reabsorbtion by the
kidney and is also known as the antidiuretic hormone. [0028] iii.
ISO: isoproterenol, a synthetic beta-adrenergic receptor agonist
which controls peripheral vasoconstriction, bronchodilation and
increased cardiac rate, contractility and output. [0029] iv. SNC80:
4[(.sup..alpha.R)-.alpha.-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3--
methoxybenzyl]-N,N-diethylbenzamide. An agonist of the delta-opioid
receptor that possesses anti-nociceptive action. [0030] v. PAF:
platelet-activating factor; a hormone that regulates platelet
aggregation. It is an agonist of the PAF receptor. [0031] vi.
hRANTES: human RANTES (regulated upon activation, normal T cell
expressed and secreted) is a chemoattractant for monocytes and T
cells. It is an agonist of the chemokine receptors: CCR1, CCR3,
CCR5 and GPR75. [0032] vii. Wnt5a: Ligand for members of the
frizzled family of seven transmembrane receptors. [0033] viii.
IGF1: insulin-like growth factor 1 (also known as somatomedin C), a
hormone homologous to proinsulin. [0034] ix. TGF-.beta.1:
Transforming Growth Factor-beta1, a multifunctional peptide that
controls proliferation, differentiation, and other functions in
many cell types. Many cells synthesize TGF-beta 1 and essentially
all of them have specific receptors for this peptide. TGF-beta 1
regulates the actions of many other peptidic growth factors and
determines a positive or negative direction of their effects.
[0035] b) Inverse agonist: a ligand that produces an effect
opposite to that of an agonist by occupying the same receptor.
Examples include: [0036] i. SR121463: SR121463 is a selective,
orally active, non-peptide antagonist of vasopressin (AVP) V2
receptors, with powerful aquaretic properties in various animal
species and humans. SR121463 also behaves as an inverse agonist in
cells expressing constitutively active human V2 receptor. [0037] c)
Antagonist: a ligand that counteracts the effect of another ligand
(agonist or inverse agonist) acting on a receptor by binding to the
same receptor, thus blocking or dampening the ability of the
agonist to bind (also called competitive antagonist). Neutral
antagonists have affinity but no efficacy for their cognate
receptors. [0038] d) Allosteric regulator: a ligand that modulates
receptor activity through binding at a site that is different from
that bound by orthosteric ligands (i.e. endogenous ligands).
Allosteric regulators may have an antagonistic or agonistic
effect.
[0039] Chromophore: A small molecule, or a part of a larger
molecule, that is responsible for the spectral band of the
molecule.
[0040] Biosensor: A type of biomolecular probe that measures the
presence or concentration of biological molecules, biological
structures, activity state etc., by translating a biochemical
interaction at the probe surface into a quantifiable physical
signal such as light or electric pulse.
[0041] Receptor: A popular and generally accepted hypothesis that
appears to explain many pharmacodynamic phenomena holds that
specialized protein molecules on the surfaces of cells provide a
"fit" for an intrinsic molecule (such as a hormone or
neurotransmitter) or a drug such that when that molecule occupies
(binds to) that area, it leads to a biochemical or physiologic
response. This idea is often compared to the operation of a lock
(receptor) by a key (ligand). Examples of GPCR receptors include:
[0042] a) .beta..sub.2-AR: beta-2 adrenergic receptor [0043] b)
Frizzled 4 (Fz4): a seven transmembrane receptor that selectively
recognizes hormones of the Wnt family. [0044] C) V2R: Vasopressin
V2 receptor [0045] d) V1aR: Vasopressin V1a receptor [0046] e)
.delta.-OR: 5-opioid receptor [0047] f) PAFR: platelet-activating
factor receptor [0048] g) CCR5: CC chemokine receptor type 5 [0049]
h) AT1aR: angiotensin receptor type 1a
[0050] Signalling molecule: a membrane or soluble protein involved
in the transaction of signals in cells initiated by hormones,
neurotransmitters or synthetic ligands.
[0051] Identity as known in the art, is a relationship between two
or more polypeptide sequences, as determined by comparing the
sequences. In the art, "identity" also means the degree of sequence
relatedness between polypeptides as determined by the match between
strings of such sequences. "Identity" and "similarity" can be
readily calculated by known methods, including, but not limited to,
those described in (Computational Molecular Biology, Lesk, A. M.,
Ed., Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, Smith, D. W., Ed., Academic Press,
New York, 1993; Computer Analysis of Sequence Data, Part I,
Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey,
1994; Sequence Analysis in Molecular Biology, von Heinje, G.,
Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
and Devereux, J., Eds., M Stockton Press, New York, 1991; and
Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073
(1988).
[0052] By way of example, a polypeptide sequence may be identical
to the reference sequence, that is be 100% identical, or it may
include up to a certain integer number of amino acid residue
alterations as compared to the reference sequence such that the %
identity is less than 100%. Such alterations are selected from: at
least one conservative or non-conservative amino acid residue
substitution, deletion, or insertion, and wherein said alterations
may occur at the amino- or carboxy-terminal positions of the
reference polypeptide sequence or anywhere between those terminal
positions, interspersed either individually among the amino acid
residues in the reference sequence or in one or more contiguous
groups within the reference sequence. The number of amino acid
residue alterations for a given % identity is determined by
multiplying the total number of amino acids in the reference
polypeptide by the numerical percent of the respective percent
identity (divided by 100) and then subtracting that product from
said total number of amino acids in the reference polypeptide.
[0053] Conservative amino acid variants can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methyl-glycine, allothreonine,
methylthreonine, hydroxy-ethylcysteine, hydroxyethylhomocysteine,
nitro-glutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenyl-alanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is carried out in a cell-free system
comprising an E. coli S30 extract and commercially available
enzymes and other reagents. Proteins are purified by
chromatography. (Robertson, et al., J. Am. Chem. Soc, 113:
2722,1991; Ellman, et al., Methods Enzymol, 202: 301, 1991; Chung,
et al., Science, 259: 806-9, 1993; and Chung, et al, Proc. Natl.
Acad. Sci. USA, 90: 10145-9, 1993). In a second method, translation
is carried out in Xenopus oocytes by microinjection of mutated mRNA
and chemically aminoacylated suppressor tRNAs (Turcatti, et al, J.
Biol. Chem., 271: 19991-8, 1996). Within a third method, E. coli
cells are cultured in the absence of a natural amino acid that is
to be replaced (e.g., phenylalanine) and in the presence of the
desired non-naturally occurring amino acid(s) (e.g.,
2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or
4-fluorophenylalanine). The non-naturally occurring amino acid is
incorporated into the protein in place of its natural counterpart.
(Koide, et al, Biochem., 33: 7470-6, 1994). Naturally occurring
amino acid residues can be converted to non-naturally occurring
species by in vitro chemical modification. Chemical modification
can be combined with site-directed mutagenesis to further expand
the range of substitutions (Wynn, et al. Protein Sci., 2: 395-403,
1993).
[0054] Variant: refers to a polypeptide or polynucleotide that
differs from a reference polypeptide or polynucleotide, but retains
essential properties. A typical variant of a polypeptide differs in
amino acid sequence from another, reference polypeptide. Generally,
differences are limited so that the sequences of the reference
polypeptide and the variant are closely similar overall and, in
many regions, identical. A variant and reference polypeptide may
differ in amino acid sequence by one or more modifications (e.g.,
substitutions, additions, and/or deletions). A variant of a
polypeptide includes conservatively modified variants. A
substituted or inserted amino acid residue may or may not be one
encoded by the genetic code. A variant of a polypeptide may be
naturally occurring, such as an allelic variant, or it may be a
variant that is not known to occur naturally.
[0055] Modifications and changes can be made in the structure of
the polypeptides of this disclosure and still obtain a molecule
having similar characteristics as the polypeptide (e.g., a
conservative amino acid substitution). For example, certain amino
acids can be substituted for other amino acids in a sequence
without appreciable loss of activity. Because it is the interactive
capacity and nature of a polypeptide that defines that
polypeptide's biological functional activity, certain amino acid
residue substitutions can be made in a polypeptide sequence and
nevertheless obtain a polypeptide with like properties.
[0056] In one aspect, such variants have at least 60%, at least
70%, at least 80%, at least 85%, at least 90%, at least 91, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% sequence identity with
the reference polypeptide or polynucleotide.
Arrestin:
[0057] A receptor could either be constitutively active or inactive
and, ligands such agonists, inverse agonists and allosteric
modulators are known to modulate this activity. The interaction of
arrestins, including .beta.-arrestin (.beta.arr), with receptors,
such as but not limited to GPCRs, is a reflection of the receptor
activity. The beta-arrestins belong to the family of arrestins. It
is generally accepted that there are 4 arrestins in mammals:
arrestins 1-4. Arrestin-1 and arrestin-4 are visual arrestins
whereas arrestin-2 and arrestin-3 are widely distributed in all
tissues and correspond to beta-arrestin-1 and beta-arrestin-2
respectively. The interaction of .beta.-arrestins with receptors or
other proteins, such as a beta2-adaptin, has an impact on the
conformation of the .beta.-arrestins. This change in conformation
is linked to its property of interacting with effectors of
signaling pathways and receptor endocytosis. This characteristic of
arrestin is conserved throughout evolution of eukaryotic organisms
and, is the basis for the unimolecular RET-based conformational
sensors: .beta.-arrestin1 (also known as Arrestin-2 or
.beta.-arrestin) and .beta.-arrestin 2 (also known as Arrestin-3)
double brilliance sensors to monitor receptor activity. In this
invention, a conformational change in .beta.-arrestin is monitored
through a modulation of the RET signal.
[0058] The arrestins exemplified in the present invention are human
.beta.-arrestins (h.beta.arr) and mutants and variants thereof;
however, other arrestins are contemplated, including proteins
having at least 60%, at least 70%, at least 80%, at least 85%, at
least 90%, at least 92%, at least 94%, at least 96%, at least 98%,
or at least 99% sequence identity with h.beta.arr1, h.beta.arr2, or
with another arrestin, wherein such proteins interact with
GPCRs.
Resonance Energy Transfer Assays:
[0059] Resonance energy transfer (abbreviated RET, and also
referred to as Forster resonance energy transfer) is a mechanism
describing energy transfer between two chromophores, having
overlapping emission/absoprtion spectra. When the two chromophores
(the "donor" and the "acceptor"), are within 10-100 .ANG.
(Angstroms) of one another and their transition dipoles are
appropriately oriented, the donor chromophore is able to transfer
its excited-state energy to the acceptor chromophore through
non-radiative dipole-dipole coupling.
[0060] Bioluminescence Resonance Energy Transfer (BRET) Assay is a
proximity assay based on the non-radiative transfer of energy
between a donor bioluminophore (bioluminescent enzyme (ex:
luciferase)) and an acceptor fluorophore (ex: GFP or YFP).
[0061] As used herein, BRET1 uses coelenterazine h as the
luciferase substrate (i.e. bioluminescent initiator molecule) and
YFP and its variants as the energy acceptor. BRET2 uses
coelenterazine 400a (Perkin-Elmer, Wellesley, Mass., USA and,
Biotium Inc, Hayward, Calif., USA) as the luciferase substrate and
CFP, GFP2, GFP10, Tsapphire or mAmetrine as the energy acceptor.
BRET1 and BRET2 represent different variants of BRET that are based
on the use of different, luminescent enzymes, luciferase substrates
and different fluorescent proteins. The difference between the
BRET1 and BRET2 biosensors as used herein also incorporates
differences in both the linkers used to join the chromophores to
the beta arrestin molecules and the orientation of the chromophores
relative to each other (i.e. to which terminal of beta-arrestin are
they linked.
[0062] Each version of BRET typically uses a different
coelenterazine to be able to excite the acceptor at different
wavelengths. Typically, the acceptor for BRET1 is a YFP and for
BRET3 is an OFP (Abhijit De, Pritha Ray , Andreas Markus Loening
and Sanjiv Sam Gambhir, BRET3: a red-shifted bioluminescence
resonance energy transfer (BRET)-based integrated platform for
imaging protein-protein interactions from single live cells and
living animals The FASEB Journal. 23(8): 2702-2709, incorporated by
reference herein) For BRET2 the acceptor is typically any
fluorophore that can be excited close to 400 nM such as BFP, Cyan,
GFP or mKeima (RFP).
[0063] (i) bioluminophore: The bioluminophore in the BRET assay is
a protein, that catalyzes the reaction of a substrate (i.e. a
bioluminescent initiator molecule) producing bioluminescence.
[0064] Luciferase is an example of a protein that catalyzes the
oxidation of its substrate (ex: coelenterazine) producing light,
and can be used as a bioluminophore. As used herein, luciferases
refer to an enzyme that catalyzes a bioluminescent reaction (a
reaction that produces bioluminescence). In representative
embodiments, the subject luciferase polypeptides are polypeptide
sequences of the Renilla reniformis wild-type and mutant
luciferases, which are known and reported in Lorenz et al., Proc.
Natl. Acad. Sci. USA (1991) 88:4438-4442, Loening et al., Protein
Eng Des Sel. (2006) 19(9):391-400, and also reported in U.S. Pat.
No. 6,451,549 as SEQ ID NOS: 1 and 2, and in U.S. Pat. No.
7,842,469, the disclosure of which is herein incorporated by
reference.
[0065] In representative embodiments, the subject luciferase
polypeptides may also be mutants (also referred to as variants
herein) of wild-type luciferases found in Renilla species (e.g.,
Renilla koellikeri; Renilla muelleri and Renilla reniformis, where
in representative embodiments, the mutant luciferase is a mutant of
the Renilla reniformis wild-type luciferase). The term "mutant" is
employed broadly to refer to a protein that differs in some way
from a reference wild-type protein, where the subject protein
retains at least one biological property of the reference wild-type
(e.g., naturally occurring) protein. The term "biological property"
of the subject proteins includes, but is not limited to, spectral
properties, such as emission maximum, quantum yield, and brightness
(e.g., as compared to the wild-type protein or another reference
protein such as firefly luciferase from P. pyralis), and the like;
in vivo and/or in vitro stability (e.g., half-life); and the like.
In particular, the mutants (or variants) retain luciferase activity
(e.g., catalyze the conversion of a coelenterazine substrate into a
luminescent product in the presence of molecular oxygen). Mutants
of the disclosure include single amino acid changes (point
mutations), deletions of one or more amino acids (point-deletions),
N-terminal truncations, C-terminal truncations, insertions, and the
like.
[0066] For purposes of the disclosure, a naturally occurring
luciferase is a reference wild type luciferase for a given mutant
if the amino acid sequences of the wild-type and the mutant have
high identity over at least the length of the mutant (e.g., at
least about 90%, at least about 95%, at least about 97%, at least
about 98%, at least about 99% or higher) but will not have complete
sequence identity in representative embodiments.
[0067] In representative embodiments, the mutants encoded by the
subject polynucleotides exhibit increased light output as compared
to their corresponding reference wild-type protein. Specifically,
the subject mutants have at least enhanced light output with a
given coelenterazine substrate as compared to their corresponding
reference wild type. For purposes of the present disclosure,
increased light output is determined by evaluating at least one of
the kinetics and quantum yield of a given mutant using a convenient
assay known to those of skill in the art. In representative
embodiments in which the subject polynucleotides encode a mutant of
Renilla luciferase that exhibits enhanced light output, the encoded
mutant may include a substitution at least one of the following
positions: C124; K136; M185, and S287. In one aspect the Renilla
luciferase mutant has the following substitutions: C124A and M185V.
In another aspect the Renilla luciferase mutant has the following
substitutions: A55T, C124A and M185V, and is referred to herein as
RlucII. These mutations and variations thereof are known (see
Loening et al., Protein Eng Des Sel. (2006) 19(9):391-400 and U.S.
Pat. No. 7,842,469, both of which are incorporated herein), and are
contemplated for use in the present invention. Examples of Renilla
luciferase proteins contemplated herein include proteins that have
an amino acid-sequence selected from:
TABLE-US-00001 Rluc WT
MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIF
LHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHY
KYLTAWFELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVV
DVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVETMLPSKIMRKLEP
EEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLR
ASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDE
MGKYIKSFVERVLKNEQ* Rlucll (A55T/C124A/M185V)
MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIF
LHGNATSSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHY
KYLTAWFELLNLPKKIIFVGHDWGAALAFHYSYEHQDKIKAIVHAESVV
DVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVETVLPSKIMRKLEP
EEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLR
ASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDE
MGKYIKSFVERVLKNEQ* Rluc8 (A55T/C124A/S130A/K136R/A143M/M185V/M253L/
S287L) MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIF
LHGNATSSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHY
KYLTAWFELLNLPKKIIFVGHDWGAALAFHYAYEHQDRIKAIVHMESVV
DVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVETVLPSKIMRKLEP
EEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLR
ASDDLPKLFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFLQEDAPDE
MGKYIKSFVERVLKNEQ
portions thereof, mutants thereof, variants thereof, or
conservative variants thereof. Other luciferase variants known in
the art include those disclosed in US 2009/0136998, incorporated by
reference herein.
[0068] In representative embodiments, the mutant luciferase
polynucleotides encoded by the nucleic acids are mutants of
luciferase polynucleotides that employ a coelenterazine as a
substrate, where the term coelenterazine refers collectively to
native coelenterazine, as well as analogues thereof, where
representative coelenterazine analogues of interest include, but
are not limited to: benzyl-coelenterazine; coelenterazine-cp;
coelenterazine-n; bis-deoxy-coelenterazine (also known as
coelenterazine 400a and DeepBlue-coelenterazine); and the like.
[0069] In addition to the above-described specific subject
polynucleotide compositions, also of interest are homologues of the
above-sequences. With respect to homologues of the subject
polynucleotide, the source of homologous genes may be any species
of plant or animal, or the sequence may be wholly or partially
synthetic. In certain embodiments, sequence similarity between
homologues is at least about 20%, at least about 25%, and may be
30%, 35%, 40%, 50%, 60%, 70% or higher, including 75%, 80%, 85%,
90% and 95% or higher. Sequence similarity is calculated based on a
reference sequence, which may be a subset of a larger sequence,
such as a conserved motif, coding region, flanking region, and the
like. A reference sequence will usually be at least about 18 nt
long, more usually at least about 30 nt long, and may extend to the
complete sequence that is being compared. Algorithms for sequence
analysis are known in the art, such as BLAST, described in Altschul
et al. (1990), J. Mol. Biol. 215:403-10 (using default settings,
e.g. parameters w=4 and T=17). The sequences provided herein are
used for recognizing related and homologous nucleic acids in
database searches.
[0070] (ii) fluorophore: The fluorophore in the BRET assay is a
fluorescent protein.
[0071] Green fluorescent protein ("GFP") is a 238 amino acid
residues polypeptide with amino acid residues 65 to 67 involved in
the formation of the chromophore, which does not require additional
substrates or cofactors to fluoresce (see, e.g, Prasher et al,
1992, Gene 111:229-233; Yang et al, 1996, Nature Biotechnol.
14:1252-1256; and Cody et al, 1993, Biochemistry 32:1212-1218).
Thus, in one embodiment, such a fluorophore is a green fluorescent
protein (GFP) (referring to native Aequorea green fluorescent
protein), and variants thereof.
[0072] A broad range of fluorescent protein genetic variants have
been developed over the past several years that feature
fluorescence emission spectral profiles spanning almost the entire
visible light spectrum. Such variants of the GFP gene have been
found useful to enhance expression and to modify excitation and
fluorescence. Extensive mutagenesis efforts in the original
jellyfish protein have resulted in fluorescent probes that range in
color from blue to yellow. For example, substitution of a serine at
position 65 to either alanine, glycine, isoleucine, or threonine
results in mutant GFPs with a shift in excitation maxima and
greater fluorescence than wild type protein when excited at 488 nm
(see, e.g, Heim et al, 1995, Nature 373:663-664; U.S. Pat. No.
5,625,048; Delagrave et al, 1995, Biotechnology 13:151-154;
Cormacketal, 1996, Gene 173:33-38; and Cramer et al, 1996, Nature
Biotechnol. 14:315-319). Longer wavelength fluorescent proteins,
emitting in the orange and red spectral regions, have been
developed from the marine anemone Discosoma striata and reef corals
belonging to the class Anthozoa. Still other species produce
similar proteins having cyan, green, yellow, orange, red, and
far-red fluorescence emission. Thus, in another embodiment, GFPs
are isolated from organisms other than the jellyfish, such as, but
not limited to, the sea pansy, Renilla reriformis, or are variants
thereof.
[0073] Thus, a fluorophore, as used herein, includes wild type
green fluorescent protein and its variants, as well as fluorescent
proteins and variants from other species. Such fluorophores are
many, and are known to those of skill in the art. They include, but
are not limited to: [0074] Green Fluorescent Proteins include GFP
(wt), EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP,
TurboGFP, AcGFP, ZsGreen, T-Sapphire. [0075] Blue Fluorescent
Proteins include Blue Fluorescent Protein (BFP), EBFP, EBFP2,
Azurite, GFP2, GFP10, and mTagBFP; [0076] Cyan Fluorescent Proteins
include Cyan Fluorescent Protein (CFP), ECFP, mECFP, Cerulean,
CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mCFPm.sup.m, and mTFP1
(Teal); [0077] Yellow Fluorescent Proteins include Yellow
Fluorescent Protein (CFP), EYFP, Topaz, Venus, mCitrine, YPet,
TagYFP, PhiYFP, ZsYellow1, and mBanana; [0078] Orange Fluorescent
Proteins include Orange Fluorescent Protein (OFP), Kusabira Orange,
Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem,
TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express (T1), DsRed-Monomer,
and mTangerine; and [0079] Red Fluorescent Proteins include Red
Fluorescent Protein (RFP), mRuby, mApple, mStrawberry, AsRed2,
mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem,
HcRed-Tandem, mPlum, tdTomato, and AQ143.
[0080] Both green and yellow fluorescent proteins have been
genetically engineered to create circular permutations of the
original sequences that enable fusions to amino acids far removed
from the normal amino and carboxy termini (abbreviated cpGFP and
cpYFP).
[0081] The choice of a suitable fluorophore for use in a BRET assay
will be known to one of skill in the art. In one embodiment,
fluorophores include green fluorescent protein--wild type (GFP-wt),
yellow fluorescent protein (YFP), Venus, Topaz, ZsYellow1,
mOrange2, mKeima, blue fluorescent protein (BFP), cyan fluorescent
protein (CFP), Tsapphire, mAmetrine, green fluorescent protein-2
(GFP2) and green fluorescent protein-10 (GFP10), or variants
thereof. Fluorescent proteins having an excitation peak close to
400 nm may be particularly suitable. More particular examples of
fluorophores include mAmetrine, cyan fluorescent protein (CFP), and
GFP10.
[0082] Fluorescence Resonance Energy Transfer (FRET) Assay. Similar
to BRET, FRET involves the transfer of energy from an excited donor
fluorophore to an adjacent acceptor fluorophore. For example, CFP
and YFP, two color variants of GFP, can be used as donor and
acceptor, respectively.
[0083] (i) fluorophore: The fluorophores in the FRET assay are
fluorescent proteins, having the same properties as the fluorophore
as defined above for the BRET assay.
[0084] Two fluorophores are employed in FRET, one as donor and one
as acceptor. The term "donor fluorophore-acceptor pair," as used
herein, means a donor fluorophore and an acceptor that has an
absorbance spectrum overlapping the emission spectrum of the donor
fluorophore. Where the first member of the pair is a donor
fluorophore, the second member of the pair will be an acceptor.
Where the first member of the pair is an acceptor, the second
member of the pair will be a donor fluorophore.
[0085] Any of a number of fluorophore combinations can be selected
for use in the FRET embodiment of the present invention (see for
example, Pesce et al., eds, Fluorescence Spectroscopy, Marcel
Dekker, New York, 1971; White et al., Fluorescence Analysis: A
practical Approach, Marcel Dekker, New York, 1970; Handbook of
Fluorescent Probes and Research Chemicals, 6th Ed, Molecular
Probes, Inc., Eugene, Oreg., 1996; which are incorporated herein by
reference). In general, a preferred donor fluorophore is selected
that has a substantial spectrum of the acceptor fluorophore.
Furthermore, it may also be desirable in certain applications that
the donor have an excitation maximum near a laser frequency such as
Helium-Cadmium 442 nM, Argon 488 nM, Nd:YAG 532 nm, He--Ne 633 nm,
etc. In such applications the use of intense laser light can serve
as an effective means to excite the donor fluorophore. In certain
preferred embodiments, the acceptor fluorophore has a substantial
overlap of its excitation spectrum with the emission spectrum of
the donor fluorophore. In some cases, the wavelength maximum of the
emission spectrum of the acceptor moiety is preferably at least 10
nm greater than the wavelength maximum of the excitation spectrum
of the donor moiety. Additional examples of useful FRET labels
include, e.g., those described in U.S. Pat. Nos. 5,654,419,
5,688,648, 5,853,992, 5,863,727, 5,945,526, 6,008,373, 6,150,107,
6,177,249, 6,335,440, 6,348,596, 6,479,303, 6,545,164, 6,849,745,
6,696,255, and 6,908,769 and Published U.S. Patent Application Nos.
2002/0168641, 2003/0143594, and 2004/0076979, the disclosures of
which are incorporated herein by reference.
[0086] As indicated above, the donor and acceptor fluorophores
should be capable of forming a FRET pair. Many suitable fluorophore
pairs are familiar to those of skilled in the art. In one
embodiment, the FRET pair comprises one of CFP-YFP, GFP-mRFP1,
YFP-mRFP1, GFP-RFP, sCFP3A-sYFP2, as well as sCFP3A in combination
with circular permutations of sYFP2 (such as cp145, cp173, and
cp229).
[0087] Circular permutations can be made by PCR. Such permutations
have been described (see U.S. Pat. No. 6,699,687 and Takeharu
Nagai, Shuichi Yamada, Takashi Tominaga, Michinori Ichikawa and
Atsushi Miyawaki (2004) PNAS101(29): 10554-10559, incorporated by
reference herein). They create variants of a FRET sensor with
different orientations of the donor vs acceptor's chromophore.
Linkers
[0088] The chromophores are each attached to the beta-arrestin
molecule through independent linkers. Linkers may be employed to
provide the desired conformation of the BRET/FRET label
chromophores within the labeled compound, e.g., including the
separation between chromophores in a BRET/FRET pair. The linkers
may be bound to the C-terminal, the N-terminal, or at an
intermediate position.
[0089] In one embodiment, the linkers are peptide linkers,
typically ranging from 2 to 30 amino acids in length. The
composition and length of each of the linkers may be chosen
depending on various properties desired such as flexibility and
aqueous solubility. For instance, the peptide linker may comprise
relatively small amino acid residues, including, but not limited
to, glycine; small amino acid residues may reduce the steric bulk
and increase the flexibility of the peptide linker. The peptide
linker may also comprise polar amino acids, including, but not
limited to, serine. Polar amino acid residues may increase the
aqueous solubility of the peptide linker. Furthermore, programs
such as Globplot 2.3 (http://globplot.embl.de/cgiDict.py), may be
used to help determine the degree of disorder and globularity, thus
also their degree of flexibility.
[0090] By way of example, contemplated linkers include:
GDLRRALENSHASAGYQACGTGS and CLEDPRVPVAT. Short and relatively
flexible linkers include GSAGT and KLPAT. Longer 15-residues long
linkers, such as GSAGTGSAGTGSAGT (=3.times.GSAGT linker) and
KLPATKLPATKLPAT (=3.times.KLPAT linker) are also contemplated;
these latter 2 linkers are predicted (Globplot 2.3:
http://globplot.embl.de/cgiDict.py) to be disordered and
non-globular sequences, and thus flexible. Alpha-helix structured
rigid linker, REAAAREAAAREAAAR (16-residues long), is also
contemplated.
[0091] The linkers may be attached to the beta-arrestin at the
N-terminus, the C-terminus, or between the two termini of the
beta-arrestin. When attaching in between the two termini, the
linker may be attached, for instance to the first or second loop of
the beta-arrestin.
Methods
[0092] Expression vectors. Plasmids encoding Flag-AT1aR, CCR5
(Pleskoff et al, 1997) and Myc-PAFR (Marrache et al, 2002) were
provided by S. Meloche, N. Heveker and S. Chemtob, respectively
(Universite de Montreal, Quebec, Canada) and WT .beta.-arr2 was a
generous gift from S. Marullo (Institut Cochin, Paris). Myc-V2R and
HA-V1 aR (Terrillon et al, 2003), Myc-.beta.2-AR (Hebert et al,
1996), Myc-.delta.-OR (Petaja-Repo et al, 2002), V2R-GFP (Charest
& Bouvier, 2003), .beta.2-AR-GFP (Mercier et al, 2002).
[0093] BRET1 biosensors: .beta.-arr2-YFP (Angers et al, 2000) and
Luc-.beta.-arr2 (Perroy et al, 2003) have been described
previously. Luc-.beta.-arr-YFP was generated by subcloning the
coding sequence of enhanced YFP in-frame at the C terminus of
.beta.-arr2 in pcDNA3.1-Luc-.beta.-arr2, yielding
Luc-.beta.-arr-YFP with flexible spacers of 23 aa between Luc and
.beta.-arr, and 10 aa between .beta.-arr and YFP. Mutation of
arginine 169 into glutamate in Luc-.beta.-arr (R169E)-YFP was
generated by PCR site-directed mutagenesis using
Luc-.beta.-arr-YFP. It should be noted that while the construct
described here is specific for Luc-.beta.-arr-YFP, a construct
leading to the production of a YFP-.beta.-arr-Luc biosensor is
feasible. Moreover, the resulting biosensor, YFP-.beta.-arr-Luc,
would be expected to function in the same manner as
Luc-.beta.-arr-YFP. Similarly, DNA constructs may be devised for
the specific expression of Luc-.beta.-arr-GFP, GFP-.beta.-arr-Luc
biosensors, and variants thereof.
[0094] BRET2 biosensors: Acceptor-beta-arr1/2-RlucII, with Acceptor
being either mAmetrine, sCFP3A or GFP10, were derived from
previously published GFP10-EPAC-RlucII fusion protein (Leduc et al.
JPET 2009) by excising the EPAC coding sequence with Acc651-HindIII
restriction enzymes and replacing it with a PCR-amplified coding
sequence of human beta-arrestin1 or beta-arrestin2. Sequence
integrity was confirmed by DNA sequencing.
[0095] Cell culture. Human embryonic kidney 293 (HEK293) cells and
simian kidney fibroblast (COS) cells were maintained as described
previously (Charest & Bouvier, 2003). Cells were transfected
with the indicated plasmids using the calcium phosphate
precipitation method (Sambrook et al, 1989) or the FuGENE 6
transfection reagent (Roche Applied Science, Laval, Canada)
according to the manufacturer's protocol. The experiments were
performed 48 h after transfection.
[0096] Fluorescence microscopy. To detect Myc-.beta.2-AR and
Myc-V2R, cells were incubated with anti-Myc 9E10 monoclonal
antibody (ascite fluid from our core facility) for 1 h at 4.degree.
C. and then treated with the appropriate agonist (Sigma, Oakville,
Canada) for 2 or 30 min at 37.degree. C. Cells were then fixed and
permeabilized before adding Texas-red-conjugated secondary antibody
(Santa Cruz Biotechnology Inc., Santa Cruz, Calif., USA). The
samples were analysed by confocal laser-scanning microscopy using a
Leica TCS SP1. Measurements were as follows: YFP (green),
.lamda.ex=488 nm, .lamda.em=540/25 nm; Texas red (red),
.lamda.ex=568 nm, .lamda.em=610/30 nm.
[0097] BRET assays. Assessment of .beta.-arr recruitment in BRET
was performed as described previously (Charest & Bouvier,
2003). Briefly, cells were distributed in 96-well microplates
(Corning, Corning, USA) and incubated with or without agonist for
the indicated time at 25.degree. C. The appropriate Luc substrate
was added to a final concentration of 5 mM, either simultaneously
with the agonist (time course) or following agonist treatment
(single measurement or dose dependency), and readings were
collected using a Multilabel Reader Mithras LB 940 (Berthold
Technologies, Bad Wildbad, Germany). To detect BRET1 between Luc
and YFP, coelenterazine h (Molecular Probes, Burlington, Canada)
was used as substrate and light emission was detected at
approximately 460-500 nm (Luc) and approximately 510-550 nm (YFP),
whereas for BRET2 detection (Luc and GFP), coelenterazine 400a
(Perkin-Elmer, Wellesley, Mass., USA or Biotium Inc, Hayward,
Calif., USA) and filters at approximately 330-470 nm (Luc) and
approximately 495-535 nm (GFP2/GFP10) were used. (Broadly speaking,
ranges for the detection of light emission for BRET1 are
approximately 440-510 nm (Luc) and 510-570 nm (YFP), while those
for BRET2 are approximately 320-490 nm (Luc) and 490-550 nm (GFP)).
Those for BRET3 are ????. The BRET signal was determined by
calculating the ratio of the light emitted by the fluorescent
acceptor and the light emitted by Luc. The values were corrected by
subtracting the background BRET signals detected when
Luc-.beta.-arr was expressed alone. Expression levels of the
different receptors transfected were verified by enzyme-linked
immunosorbent assay (ELISA) (Charest & Bouvier, 2003).
[0098] Receptor endocytosis assay. Receptor endocytosis was
measured by ELISA as described previously (Charest & Bouvier,
2003).
[0099] Z'-factor determination. HEK293T cells were cultured in DMEM
supplemented with 10% fetal bovine serum, 100 units/ml penicillin
and streptomycin (Wisent Inc). 3.0.times.106 cells were seeded in
10 cm dishes. Transient transfection was performed using
polyethyleneimine (PEI; Polysciences) at a DNA:PEI ratio. 24 h
post-transfection, cells were detached, seeded in pretreated
poly-L-ornithine hydrobromide (Sigma-Aldrich) 96-well white plates
at 50,000 cells per well, and re-incubated at 37.degree. C. for an
additional 24 h before being processed. Cells were washed once with
Tyrode's buffer directly in the 96-well plates and incubated in
buffer with or without 100 nM of AVP for 25 to 35 min.
Coelenterazine 400A was added to a final concentration of 5 .mu.M
in Tyrode's buffer 5 min before reading. Readings were collected as
a sequential integration of the signals detected in the 480.+-.20
and 530.+-.20 nm window for the RlucII Renilla luciferase and GFP10
light emissions, respectively. The BRET signal was determined by
calculating the ratio of the light intensity emitted by the GFP10
over the light intensity emitted by the RLucII.
Results
[0100] Double-brilliance .beta.-arr sensor (BRET1): Inspired by
previous reports of intramolecular fluorescence resonance energy
transfer (FRET)-based biosensors (Zhang et al, 2002) showing that
resonance energy transfer (RET) is sensitive to changes in the
relative positions of the donor and acceptor molecules, the
feasibility of monitoring whether conformational changes of
.beta.-arr using an intramolecular BRET approach was assessed. A
double-brilliance .beta.-arr was engineered in which Luc was fused
to the N terminus of .beta.-arr2 and YFP to its C terminus,
yielding Luc-.beta.-arr-YFP (FIG. 1). To test the functionality of
Luc-.beta.-arr-YFP, the ability of this molecule to be recruited to
agonist-stimulated class A (receptors interacting transiently with
.beta.arr) .beta.2-adrenergic receptor (.beta.2-AR) and class B
(receptors interacting stably with .beta.arr) V2 vasopressin
receptor (V2R) by fluorescence microscopy was determined. As shown
in FIG. 2A, agonist stimulation led to rapid translocation of
Luc-.beta.-arr-YFP to the plasma membrane, colocalizing with
Myc-tagged .beta.2-AR and V2R (Myc-.beta.2-AR; Myc-V2R). The
patterns of Luc-.beta.-arr-YFP interaction were consistent with
those observed for class A (transient .beta.-arr interaction) and B
(stable .beta.-arr association) receptors in similar experiments
using a .beta.-arr-green fluorescent protein (GFP) conjugate
(Oakley et al, 2000). Indeed, whereas Luc-.beta.-arr-YFP was
recruited to both .beta.2-AR and V2R after 2 min of stimulation, it
returned to the cytoplasm after 30 min in Myc-.beta.2-AR-expressing
cells but remained colocalized with Myc-V2R in endocytic
vesicles.
[0101] To quantitatively assess the recruitment of
Luc-.beta.-arr-YFP to agonist-activated GPCRs, an intermolecular
BRET2 assay that takes advantage of the different spectral
properties of Luc substrates that allow energy transfer to
different fluorescent acceptors (Milligan, 2004) was used.
Luc-.beta.-arr-YFP was transiently coexpressed with the receptors,
and the agonist-induced BRET2 between Luc-.beta.-arr-YFP and either
.beta.2-AR-GFP or V2R-GFP was measured in the presence of
DeepBlueC.TM. coelenterazine, allowing transfer of energy to GFP.
As shown in FIG. 2, agonist stimulation promoted a time-dependent
(FIG. 2B) and dose-dependent (FIG. 2C) increase in BRET2,
reflecting the recruitment of Luc-.beta.-arr-YFP to the receptors.
Similar kinetics and EC50 were obtained for the recruitment of both
Luc-.beta.-arr-YFP and Luc-.beta.-arr, indicating that
double-brilliance .beta.-arr is as efficiently recruited to the
receptors as the singly conjugated construct. It should be noted
that, although the maximum agonist-promoted BRET increase observed
with the class A .beta.2-AR is less than that observed with the
class B V2R, the stability of the signals was similar, indicating
that the signal observed with .beta.2-AR reflects a steady state
corresponding to constant association and dissociation of
.beta.-arr from the activated receptors.
[0102] To assess the biological activity of Luc-.beta.-arr-YFP, its
capacity to promote receptor endocytosis in COS cells, which
express low endogenous levels of .beta.-arr, was tested. As shown
in FIG. 2D, agonist-promoted .beta.2-AR and V2R endocytosis was
considerably increased when overexpressing Luc-.beta.-arr-YFP. Even
though this increase in receptor endocytosis was not as pronounced
as that obtained by the overexpression of wild-type (WT)
.beta.-arr, it suggests that Luc-.beta.-arr-YFP retains significant
biological activity.
[0103] Agonist-induced conformational changes of .beta.-arr: To
assess whether Luc-.beta.-arr-YFP could be used to monitor the
conformational rearrangement of .beta.-arr upon receptor
activation, the construct was expressed with and without V2R, and
BRET was measured in the presence of coelenterazine h, allowing
transfer of energy to YFP. As shown in FIG. 3A, an important basal
BRET signal could be measured in cells transfected with
Luc-.beta.-arr-YFP, reflecting the proximity of the energy donor
and acceptor in the construct. Arginine vasopressin (AVP)
stimulation of cells coexpressing V2R led to a significant increase
in BRET, suggesting movement of Luc and YFP relative to each other.
To rule out the possibility that this increased signal results from
intermolecular BRET between individual Luc-.beta.-arr-YFP molecules
brought together through oligomerization (Hirsch et al, 1999) or
clustering at the plasma membrane, the occurrence of BRET in cells
transiently expressing Luc-.beta.-arr and .beta.-arr-YFP was
determined. In transfection conditions leading to equivalent
fluorescence and luminescence levels as those obtained in
Luc-.beta.-arr-YFP-expressing cells, coexpression of Luc-.beta.-arr
and .beta.-arr-YFP led to the detection of only a marginal basal
BRET that could not be modulated by V2R stimulation (FIG. 3A). This
observation demonstrates that the AVP-induced increase in BRET
signal observed in cells transfected with Luc-.beta.-arr-YFP
results from a change in intramolecular BRET. As variations in RET
can reflect changes in both the distance and orientation between
the energy donor and acceptor molecules (Andrews & Demidov,
1999), the observed agonist-promoted increase in the
Luc-.beta.-arr-YFP intramolecular BRET could indicate that the N
terminus and C terminus are either brought closer or are in a more
permissive BRET orientation following activation.
[0104] To further characterize the agonist-induced change in the
conformation of .beta.-arr, the kinetics and dose dependency of
AVP-mediated BRET increase were assessed. Real-time BRET
measurements show a time-dependent AVP-induced conformational
change of .beta.-arr, with half-time of maximal BRET increase
(t1/2) of 5.1.+-.1.5 min (FIG. 3B). The kinetics are significantly
slower (P<0.02) than that of the AVP-induced recruitment of
.beta.-arr (t1/2=0.8.+-.0.2 min; FIG. 2B, right panel), suggesting
that the conformational change observed in Luc-.beta.-arr-YFP
occurs after its initial recruitment to the activated V2R. The
difference in kinetics cannot result from inter-experimental
variations because similar results were obtained when the two
events were measured in the same cell population expressing V2R-GFP
and Luc-.beta.-arr-YFP (data not shown). Despite the difference in
kinetics, the efficacy of AVP to induce a conformational change in
Luc-.beta.-arr-YFP (FIG. 3C) was similar to that observed for
.beta.-arr recruitment (FIG. 2C, right panel), indicating that
these two events are directly linked and reflect the binding
affinity of V2R for AVP (KD .about.1.times.10-9 M).
[0105] The observed kinetic lag between .beta.-arr recruitment and
its conformational change could be consistent with the proposal
that inactive .beta.-arr is first recruited to the activated GPCR
where its interaction with the GRK-phosphorylated residues
subsequently induces the release of its C-tail (Gurevich &
Gurevich, 2003). Alternatively, such a lag could indicate that the
intramolecular BRET changes observed with Luc-.beta.-arr-YFP result
from the subsequent recruitment of .beta.-arr-interacting proteins
(e.g. clathrin and AP2 or signalling proteins such as c-Src, Raf1,
ERK1/2, ASK1 and JNK3) to the receptor-bound .beta.-arr (Lefkowitz
& Whalen, 2004). Interestingly, a .beta.-arr (R169E) mutant
shown to bind to GPCRs in a phosphorylation-independent manner,
probably as a result of a constitutively open conformation (Kovoor
et al, 1999) resulted, when inserted between Luc and YFP
(Luc-.beta.-arr(R169E)-YFP), in basal and AVP-stimulated BRET
signals similar to those observed with WT Luc-.beta.-arr-YFP (FIG.
4). This indicates that the engagement of .beta.arr by the
activated receptor can be detected by the double brilliance
.beta.arr independently of the phosphorylation state of the
receptor.
[0106] A general biosensor to monitor GPCR activity: To assess
whether Luc-.beta.-arr-YFP could be used as a general GPCR activity
sensor, a determination of whether its agonist-induced
conformational change could be promoted by other receptors was
made, particularly those of class A, which are believed to interact
only transiently with .beta.-arr. Recruitment of Luc-.beta.-arr-YFP
and agonist promoted intramolecular BRET were assessed in cells
coexpressing different receptors of class A (.beta.2-AR, V1
vasopressin receptor (V1 aR), d-opioid receptor (.delta.-OR)) and
class B (platelet-activating factor receptor (PAFR), CC chemokine
receptor type 5 (CCR5), angiotensin receptor type 1a (AT1aR)). As
shown in FIG. 5A, agonist stimulation efficiently induced the
recruitment of Luc-.beta.-arr-YFP to the plasma membrane, with the
expected interaction patterns for all class A (transient) and class
B (stable) receptors. In all cases, activation of
Luc-.beta.-arr-YFP mediated by class A and B receptors was
accompanied by a significant increase in BRET (FIG. 5B).
Interestingly, although the kinetics and stability of the BRET
increase were found to be similar for receptors of class A and B
(data not shown), a tendency of class A receptors to induce smaller
BRET increases was observed. As previously noted when comparing the
BRET-detected recruitment of .beta.-arr to class A .beta.2-AR and
class B V2R (FIG. 2B), this probably indicates that the BRET assays
provide a steady-state signal reflecting continuous rounds of
association-dissociation cycles. In any case, these results suggest
that Luc-.beta.-arr-YFP can be used as a general biosensor to
monitor GPCR activity and that the interaction can be monitored for
extended periods of time making it compatible with its use in high
through put screening assays that request long lived signals. When
compared with the intermolecular BRET-based .beta.-arr recruitment
assays (Angers et al, 2000; Bertrand et al, 2002),
double-brilliance .beta.-arr avoids the difficulty of expressing
the appropriate ratio of energy donor and acceptor constructs and
allows the study of unmodified GPCRs.
[0107] The interaction of .beta.-arr with the GRK-phosphorylated
GPCRs is thought to induce the release of .beta.-arr's C-tail and
the opening of its structure (Gurevich and Gurevich 2003),
subsequently leading to the recruitment of
.beta.arrestin-interacting proteins (Lefkowitz and Whalen 2004). To
assess if the conformational change of .beta.-arr detected with the
double brilliance .beta.-arr could also be detected using .beta.arr
mutants believed to be constitutively in the open state, assessment
was made of the agonist-promoted BRET signal of two other
.beta.-arr mutants (.beta.-arr(3A): 1387A, V388A, F389A;
.beta.-arr(IV): 1387A, V388A),inserted between Luc and YFP
(Luc-.beta.arr(3A)-YFP and Luc-.beta.arr(IV)-YFP). These mutant
.beta.-arrs are believed to be constitutively active due to the
disruption of the polar core keeping .beta.arrestin in a closed and
inactive conformation (Gurevich 1998). As shown in FIG. 6, while
the basal BRET signal observed with each Luc-.beta.arr-YFP
constitutively active mutant (Luc-.beta.arr(3A)-YFP and
Luc-.beta.arr(IV)-YFP) was found to be similar to that of wild-type
Luc-.beta.arr-YFP, the agonist-induced BRET increase was
significantly reduced by the mutations (FIG. 6, inset).
[0108] In addition to agonists, the activity of ligands with
inverse agonist efficacy towards specific signalling pathways can
be detected by the double brilliance .beta.-arr. As shown in FIG.
7, the V2R inverse agonist SR121463 that inhibits cyclic AMP
production can promote an increase in the BRET signal in cells
co-expressing wild type V2R and Luc-.beta.-arr-YFP.
[0109] Double brilliance .beta.-arr may also prove to be an
effective tool in the study of the increasingly diverse roles
played by .beta.-arr, such as its involvement with receptors other
than GPCRs and diverse signaling molecules in different systems
(FIG. 8). A list of some of the proteins that have been shown to
interact with .beta.arr and which activity could be monitored by
double brilliance .beta.-arr is presented in Table 1. The spectrum
of receptors capable of utilizing .beta.-arr for endocytosis via
clathrin binding sites has significantly increased (Lefkowitz and
Whalen 2004). For example, .beta.-arr appears to be required for
engulfing Frizzled-4, an atypical seven-transmembrane domain
receptor, through interaction with the adaptor protein
Dishevelled-2 phosphorylated by PKC (Chen et al. 2003a); for the
endocytosis of receptors with serine/threonine kinase activity such
as the transforming growth factor .beta. receptor (TGF-.beta.R), in
a manner dependent on the phosphorylation of RIII by RII (Chen et
al. 2003b); as well as for the endocytosis of the IGF1 receptor, in
a manner that is independent from its phosphorylation (Dalle et al.
2001). This indicates that the .beta.arr double brillance could be
a general biosensor of the activity of many distinct receptors and
signalling molecules.
TABLE-US-00002 TABLE 1 List of proteins capable of interacting with
.beta.-arrestin Binding Protein .beta.-arrestin isoform Type of
protein Clathrin .beta.-arr 1, 2 trafficking AP2 .beta.-arr 1, 2
trafficking NSF .beta.-arr 1 trafficking ARF6 .beta.-arr 2, 1 Small
G/GEFs ARNO .beta.-arr 2 Small G/GEFs Ral-GDS .beta.-arr 1, 2 Small
G/GEFs RhoA .beta.-arr 1 Small G/GEFs MAPK cascade components:
Signaling ASK1 .beta.-arr 1, 2 c-Raf-1 .beta.-arr 1, 2 JNK3
.beta.-arr 2, 1 ERK2 .beta.-arr 1, 2 Nonreceptor tyrosine kinases:
signaling c-Src .beta.-arr 1, 2 Yes .beta.-arr 1 Hck .beta.-arr 1
Fgr .beta.-arr 1 Others: signaling Mdm2 .beta.-arr 1, 2
I.kappa.B.alpha. .beta.-arr 1, 2 PDE4D family .beta.-arr 1, 2
Dishevelled .beta.-arr 1, 2 PP2A .beta.-arr 1 (Lefkowitz &
Shenoy, 2005)
[0110] Double-brilliance .beta.-arr sensor (BRET2): In addition to
the constructs described above, the following BRET2-based
beta-arrestin 1 and 2 sensors: Acceptor (mAmetrine; sCFP3A;
GFP10)-GSAGT-.beta.Arrestin1/2-KLPAT-RlucII were also made (FIG.
9a), using similar techniques, namely:
[0111] Ametrine-h.beta.ar1r-RlucII,
[0112] Ametrine-h.beta.arr2-RlucII,
[0113] CFP-h.beta.arr1-RlucII,
[0114] CFP-h.beta.arr2-RlucII,
[0115] GFP.sup.10-h.beta.arr1-RlucII, and
[0116] GFP.sup.10-h.beta.arr2-RlucII.
[0117] An enhanced Rluc variant (Rluc II) was used with the BRET2
versions as it provides a sustained and a stronger signal with
coelenterazine 400a (200-400 times) than with the WT Rluc. The
orientation of the BRET tags and nature of the linkers in these
constructs differ from the BRET1 version. In contrast to the BRET1
sensors, these structure leads to a decrease BRET signal in
response to an agonist-promoted GPCR activation. As shown in FIGS.
9B and 2B, this inversion of the BRET signal between the BRET1 and
BRET2 versions of the beta-arrestin db sensors still lead to
similar kinetics and dose-responses to an AVP stimulation of V2R.
Both beta-arrestin1 and 2 sensors are functional and give similar
responses for the same receptor activation (FIG. 9B).
[0118] The Z'-factor is a reflection of the robustness of an assay
and should vary depending on the experimental conditions and
receptor used. With cells transiently expressing both V2R and
sensors a Z'-factor between 0.43 and 0.63 (FIG. 10) was obtained
for both BRET1 and BRET2 versions of the beta-arrestin db sensors,
providing a robust assay for monitoring GPCR activation with both
full and partial agonists (FIG. 9C). Using cell lines stably
expressing both receptor and sensor, an even better Z'-factor is
expected and thus be sufficient to develop a high throuput
screening (HTS) assay.
[0119] Since the fluorescent energy transfer of the invention is
based on stimulatory principles such as BRET, a biosensor as
described herein based on FRET instead of BRET would also be
expected to function and is included within the scope of the
present invention.
[0120] In summary, the above is believed to be the first real-time
monitoring of agonist-promoted conformational changes of .beta.-arr
in living cells using a double-brilliance .beta.-arr intramolecular
BRET-based biosensor. The conformational rearrangement of the
.beta.-arr molecule and its interaction with other proteins
reflects its transition from an inactive state to a biologically
active state that follows its initial recruitment to activated
GPCRs and involves the relative movement of the C- and N-terminus
leading to a change in the BRET signal Beta-arrestin db sensors
offer a robust assay for GPCR activation and characteristics
(unimolecular structure, ratiometric signal and recruited to most
GPCRs) that could be amenable to large-scale screening campaigns.
In conclusion, double brilliance .beta.-arr represents the first
intramolecular BRET-based biosensor that allows the monitoring of
protein conformational changes. This should lead the way to the
development of similar tools to study other proteins believed to
undergo significant conformational rearrangement linked to their
function.
[0121] Although the present invention has been described by way of
specific embodiments and examples thereof, with a particular focus
on G protein-coupled receptors, it should be noted that it will be
apparent to persons skilled in the art that modifications may be
applied to the present particular embodiment without departing from
the scope of the present invention.
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Sequence CWU 1
1
211196DNARenilla reniformisCDS(10)..(945) 1agcttaaag atg act tcg
aaa gtt tat gat cca gaa caa agg aaa cgg atg 51 Met Thr Ser Lys Val
Tyr Asp Pro Glu Gln Arg Lys Arg Met 1 5 10ata act ggt ccg cag tgg
tgg gcc aga tgt aaa caa atg aat gtt ctt 99Ile Thr Gly Pro Gln Trp
Trp Ala Arg Cys Lys Gln Met Asn Val Leu15 20 25 30gat tca ttt att
aat tat tat gat tca gaa aaa cat gca gaa aat gct 147Asp Ser Phe Ile
Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala 35 40 45gtt att ttt
tta cat ggt aac gcg gcc tct tct tat tta tgg cga cat 195Val Ile Phe
Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His 50 55 60gtt gtg
cca cat att gag cca gta gcg cgg tgt att ata cca gat ctt 243Val Val
Pro His Ile Glu Pro Val Ala Arg Cys Ile Ile Pro Asp Leu 65 70 75att
ggt atg ggc aaa tca ggc aaa tct ggt aat ggt tct tat agg tta 291Ile
Gly Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu 80 85
90ctt gat cat tac aaa tat ctt act gca tgg ttt gaa ctt ctt aat tta
339Leu Asp His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn
Leu95 100 105 110cca aag aag atc att ttt gtc ggc cat gat tgg ggt
gct tgt ttg gca 387Pro Lys Lys Ile Ile Phe Val Gly His Asp Trp Gly
Ala Cys Leu Ala 115 120 125ttt cat tat agc tat gag cat caa gat aag
atc aaa gca ata gtt cac 435Phe His Tyr Ser Tyr Glu His Gln Asp Lys
Ile Lys Ala Ile Val His 130 135 140gct gaa agt gta gta gat gtg att
gaa tca tgg gat gaa tgg cct gat 483Ala Glu Ser Val Val Asp Val Ile
Glu Ser Trp Asp Glu Trp Pro Asp 145 150 155att gaa gaa gat att gcg
ttg atc aaa tct gaa gaa gga gaa aaa atg 531Ile Glu Glu Asp Ile Ala
Leu Ile Lys Ser Glu Glu Gly Glu Lys Met 160 165 170gtt ttg gag aat
aac ttc ttc gtg gaa acc atg ttg cca tca aaa atc 579Val Leu Glu Asn
Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys Ile175 180 185 190atg
aga aag tta gaa cca gaa gaa ttt gca gca tat ctt gaa cca ttc 627Met
Arg Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe 195 200
205aaa gag aaa ggt gaa gtt cgt cgt cca aca tta tca tgg cct cgt gaa
675Lys Glu Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu
210 215 220atc ccg tta gta aaa ggt ggt aaa cct gac gtt gta caa att
gtt agg 723Ile Pro Leu Val Lys Gly Gly Lys Pro Asp Val Val Gln Ile
Val Arg 225 230 235aat tat aat gct tat cta cgt gca agt gat gat tta
cca aaa atg ttt 771Asn Tyr Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu
Pro Lys Met Phe 240 245 250att gaa tcg gat cca gga ttc ttt tcc aat
gct att gtt gaa ggc gcc 819Ile Glu Ser Asp Pro Gly Phe Phe Ser Asn
Ala Ile Val Glu Gly Ala255 260 265 270aag aag ttt cct aat act gaa
ttt gtc aaa gta aaa ggt ctt cat ttt 867Lys Lys Phe Pro Asn Thr Glu
Phe Val Lys Val Lys Gly Leu His Phe 275 280 285tcg caa gaa gat gca
cct gat gaa atg gga aaa tat atc aaa tcg ttc 915Ser Gln Glu Asp Ala
Pro Asp Glu Met Gly Lys Tyr Ile Lys Ser Phe 290 295 300gtt gag cga
gtt ctc aaa aat gaa caa taa ttactttggt tttttattta 965Val Glu Arg
Val Leu Lys Asn Glu Gln 305 310catttttccc gggtttaata atataaatgt
cattttcaac aattttattt taactgaata 1025tttcacaggg aacattcata
tatgttgatt aatttagctc gaactttact ctgtcatatc 1085attttggaat
attacctctt tcaatgaaac tttataaaca gtggttcaat taattaatat
1145atattataat tacatttgtt atgtaataaa ctcggtttta ttataaaaaa a
11962311PRTRenilla reniformis 2Met Thr Ser Lys Val Tyr Asp Pro Glu
Gln Arg Lys Arg Met Ile Thr1 5 10 15Gly Pro Gln Trp Trp Ala Arg Cys
Lys Gln Met Asn Val Leu Asp Ser 20 25 30Phe Ile Asn Tyr Tyr Asp Ser
Glu Lys His Ala Glu Asn Ala Val Ile 35 40 45Phe Leu His Gly Asn Ala
Ala Ser Ser Tyr Leu Trp Arg His Val Val 50 55 60Pro His Ile Glu Pro
Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly65 70 75 80Met Gly Lys
Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp 85 90 95His Tyr
Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys 100 105
110Lys Ile Ile Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His
115 120 125Tyr Ser Tyr Glu His Gln Asp Lys Ile Lys Ala Ile Val His
Ala Glu 130 135 140Ser Val Val Asp Val Ile Glu Ser Trp Asp Glu Trp
Pro Asp Ile Glu145 150 155 160Glu Asp Ile Ala Leu Ile Lys Ser Glu
Glu Gly Glu Lys Met Val Leu 165 170 175Glu Asn Asn Phe Phe Val Glu
Thr Met Leu Pro Ser Lys Ile Met Arg 180 185 190Lys Leu Glu Pro Glu
Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu 195 200 205Lys Gly Glu
Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro 210 215 220Leu
Val Lys Gly Gly Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr225 230
235 240Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe Ile
Glu 245 250 255Ser Asp Pro Gly Phe Phe Ser Asn Ala Ile Val Glu Gly
Ala Lys Lys 260 265 270Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly
Leu His Phe Ser Gln 275 280 285Glu Asp Ala Pro Asp Glu Met Gly Lys
Tyr Ile Lys Ser Phe Val Glu 290 295 300Arg Val Leu Lys Asn Glu
Gln305 310
* * * * *
References